Bilirubin is a reddish yellow substance found in biological substances, such as blood serum, and has the empirical formula: C.sub.33 H.sub.36 N.sub.4 O.sub.6.
Bilirubin is an end product of hemoglobin catabolism. It is conjugated with glucuronic acid in the liver, and the conjugated form is cleared from the circulation by excretion into bile. Both conjugated (direct) and unconjugated (indirect) forms of bilirubin (total bilirubin) circulate loosely bound to albumin. Conjugated bilirubin, as the glucuronide, is a mono and/or diester, reported the structures and chromogen forming reactions of which are reported in "Bilirubin Volume I" Heirwegh et al, CRC Press, 1982 at page 1237 incorporated herein by reference and is measured by direct bilirubin methods.
Elevated serum bilirubin levels are observed in a variety of conditions, including hemolytic disorders, biliary obstruction, cholestasis, hepatitis, cirrhosis, and decreased conjugation (e.g., neonatal jaundice). In obstructive jaundice, there is an increase in the total bilirubin that is primarily due to the glucuronide fraction. In hemolytic jaundice and in neonatal jaundice, the increase is primarily due to the unconjugated bilirubin fraction. In the newborn, jaundice may be caused by Rh, ABO, or other blood group incompatibilities, by hepatic immaturity, or by hereditary defects in bilirubin conjugation. Both conjugated and unconjugated bilirubin are increased in cirrhosis and hepatitis. The relative proportion of the conjugated fraction increases with progression of the disease until eventually the liver loses its ability to carry out the conjugation process.
It is apparent that while an assay for total bilirubin is important to obtain a total assessment of human condition determination of direct or conjugated bilirubin is also of significant importance.
Most bilirubin assays are based upon variations of the classical method of Van Den Bergh and Mueller in which diazotized sulfanilic acid and bilirubin, in an acidic solution, react to form azobilirubin. Accelerators and solubilizers are added to total bilirubin procedures to ensure complete reaction of unconjugated bilirubin.
The present invention is directed to a stable liquid assay composition for the quantitative determination of serum direct or conjugated bilirubin in the diagnosis and treatment of liver, hemolytic hematological, and metabolic disorders, including hepatitis and gall bladder block.